Compounds with anti-tumor activity against cancer cells bearing egfr or her2 exon 20 mutations

ABSTRACT

The present disclosure provides methods of treating cancer in a patient determined to have an EGFR and/or HER2 exon 20 mutation, such as an insertion mutation, by administering a third-generation tyrosine kinase inhibitor, such as poziotinib or afatinib.

This application is a continuation of U.S. application Ser. No.16/461,992, filed May 17, 2019, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2017/062326, filed Nov. 17, 2017, which claims the benefit of U.S.Provisional Application No. 62/423,732, filed Nov. 17, 2016, U.S.Provisional Application No. 62/427,692, filed Nov. 29, 2016, and U.S.Provisional Application No. 62/572,716, filed Oct. 16, 2017, the entirecontents of each of which are incorporated herein by reference.

The invention was made with government support under grant numberCA190628 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

This application contains a Sequence Listing XML, which has beensubmitted electronically and is hereby incorporated by reference in itsentirety. Said XML Sequence Listing, created on Aug. 22, 2022, is namedUTSCP1306USC1.xml and is 23,371 bytes in size.

BACKGROUND 1. Field of the Invention

The present invention relates generally to the field of molecularbiology and medicine. More particularly, it concerns methods of treatingpatients with EGFR and/or HER2 exon 20 mutations, such as insertionmutations.

2. Description of Related Art

Approximately 10-15% of NSCLCs harbor activating EGFR mutations. For themajority of these patients whose tumors have “classical” sensitizingmutations (L858R and exon 19 deletions), TKIs such as gefitinib anderlotinib provide dramatic clinical benefit, with approximately 70%experiencing objective responses (OR), improved progression freesurvival (PFS), and quality of life compared to chemotherapy alone(Maemondo et al., 2010). However, approximately 10-12% of EGFR mutantNSCLC tumors have an in-frame insertion within exon 20 of EGFR (Arcilaet al., 2012), and are generally resistant to EGFR TKIs. In addition,90% of HER2 mutations in NSCLC are exon 20 mutations (Mazieres et al.,2013). Together, EGFR and HER2 exon 20 mutations comprise approximately4% of NSCLC patients. The data thus far suggests that available TKIs ofHER2 (afatinib, lapatinib, neratinib, dacomitinib) have limited activityin patients with HER2 mutant tumors with many studies reporting OR ratesbelow 40% (Kosaka et al., 2017), although some preclinical activity isobserved in HER2 mouse models treated with afatinib (Perera et al.,2009).

Exon 20 of EGFR and HER2 contains two major regions, the c-helix(residues 762-766 in EGFR and 770-774 in HER2) and the loop followingthe c-helix (residues 767-774 in EGFR and 775-783 in HER2).Crystallography of the EGFR exon 20 insertion D770insNPG has revealed astabilized and ridged active conformation inducing resistance to firstgeneration TKIs in insertions after residue 764. However, modeling ofEGFR A763insFQEA demonstrated that insertions before residue 764 do notexhibit this effect and do not induce drug resistance (Yasuda et al.,2013). Moreover, in a patient derived xenograft (PDX) model of EGFR exon20 driven NSCLC where insertions are in the loop after the c-helix (EGFRH773insNPH), third generation EGFR TKIs, osimertinib (AZD9291) androciletinib (CO-1696) were found to have minimal activity (Yang et al.,2016). In a recent study of rare EGFR and HER2 exon 20 mutations, theauthors found a heterogeneous response to covalent quinazoline-basedsecond generation inhibitors such as dacomitinib and afatinib; however,concentrations required to target more common exon 20 insertionmutations were above clinically achievable concentrations (Kosaka etal., 2017). Therefore, there is a significant clinical need to identifynovel therapies to overcome the innate drug resistance of NSCLC tumorsharboring exon 20 mutations, particularly insertion mutations, in EGFRand HER2.

SUMMARY

The present disclosure provides methods and compositions for treatingcancer in patients with EGFR and/or HER2 exon 20 mutations, such as exon20 insertion mutations. In one embodiment, there is provided a method oftreating cancer in a subject comprising administering an effectiveamount of poziotinib to the subject, wherein the subject has beendetermined to have one or more EGFR exon 20 mutations, such as one ormore EGFR exon 20 insertion mutations. In particular aspects, thesubject is human.

In certain aspects, the one or more EGFR exon 20 mutations comprise oneor more point mutations, insertions, and/or deletions of 3-18nucleotides between amino acids 763-778. In some aspects, the subjecthas been determined to have 2, 3, or 4 EGFR exon 20 mutations. In someaspects, the one or more EGFR exon 20 mutations are at one or moreresidues selected from the group consisting of A763, A767, S768, V769,D770, N771, P772, and H773. In certain aspects, the subject has beendetermined to not have an EGFR mutation at residue C797. In someaspects, the one or more exon 20 mutations are selected from the groupconsisting of A763insFQEA, A767insASV, S768dupSVD, V769insASV,D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, andN771dupNPH.

In certain aspects, the subject was determined to have an EGFR exon 20mutation, such as an insertion mutation, by analyzing a genomic samplefrom the subject. In some aspects, the genomic sample is isolated fromsaliva, blood, urine, normal tissue, or tumor tissue. In particularaspects, the presence of an EGFR exon 20 mutation is determined bynucleic acid sequencing (e.g., DNA sequencing of tumor tissue orcirculating free DNA from plasma) or PCR analyses.

In certain aspects, the method further comprises administering anadditional anti-cancer therapy. In some aspects, the anti-cancer therapyis chemotherapy, radiotherapy, gene therapy, surgery, hormonal therapy,anti-angiogenic therapy or immunotherapy. In certain aspects, thepoziotinib and/or anti-cancer therapy are administered intravenously,subcutaneously, intraosseously, orally, transdermally, in sustainedrelease, in controlled release, in delayed release, as a suppository, orsublingually. In some aspects, administering the poziotinib and/oranti-cancer therapy comprises local, regional or systemicadministration. In particular aspects, the poziotinib and/or anti-cancertherapy are administered two or more times, such as daily, every otherday, or weekly.

In some aspects, the cancer is oral cancer, oropharyngeal cancer,nasopharyngeal cancer, respiratory cancer, urogenital cancer,gastrointestinal cancer, central or peripheral nervous system tissuecancer, an endocrine or neuroendocrine cancer or hematopoietic cancer,glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma,brain cancer, oropharyngeal cancer, nasopharyngeal cancer, renal cancer,biliary cancer, pheochromocytoma, pancreatic islet cell cancer,Li-Fraumeni tumors, thyroid cancer, parathyroid cancer, pituitarytumors, adrenal gland tumors, osteogenic sarcoma tumors, multipleneuroendocrine type I and type II tumors, breast cancer, lung cancer,head and neck cancer, prostate cancer, esophageal cancer, trachealcancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer,ovarian cancer, uterine cancer, cervical cancer, testicular cancer,colon cancer, rectal cancer or skin cancer. In particular aspects, thecancer is non-small cell lung cancer.

In another embodiment, there is provided a pharmaceutical compositioncomprising poziotinib for a patient determined to have one or more EGFRexon 20 mutations, such as one or more EGFR exon 20 insertion mutations.In certain aspects, the one or more EGFR exon 20 mutations comprise apoint mutation, insertion, and/or deletion of 3-18 nucleotides betweenamino acids 763-778. In certain aspects, the subject has been determinedto have 2, 3, or 4 EGFR exon 20 mutations.

In some aspects, the one or more EGFR exon 20 insertion mutations are atone or more residues selected from the group consisting of A763, A767,S768, V769, D770, N771, P772, and H773. In certain aspects, the subjecthas been determined to not have an EGFR mutation at residue C797. Inparticular aspects, the one or more exon 20 mutations are selected fromthe group consisting of A763insFQEA, A767insASV, S768dupSVD, V769insASV,D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, andN771dupNPH. In some aspects, the patient is being treated with ananti-cancer therapy.

In yet another embodiment, there is provided a method of predicting aresponse to poziotinib alone or in combination with an anti-cancertherapy in a subject having a cancer comprising detecting an EGFR exon20 mutation (e.g., EGFR exon 20 insertion mutation) in a genomic sampleobtained from said patient, wherein if the sample is positive for thepresence of the EGFR exon 20 mutation, then the patient is predicted tohave a favorable response to poziotinib alone or in combination with ananti-cancer therapy. In some aspects, the genomic sample is isolatedfrom saliva, blood, urine, normal tissue, or tumor tissue. In certainaspects, the presence of an EGFR exon 20 mutation is determined bynucleic acid sequencing or PCR analyses. In certain aspects, the EGFRexon 20 mutation comprises one or more point mutations, insertions,and/or deletions of 3-18 nucleotides between amino acids 763-778. Insome aspects, the EGFR exon 20 mutation is at residue A763, H773, A767,S768, V769, D770, N771, and/or D773. In some aspects, the EGFR exon 20mutation is selected from the group consisting of A763insFQEA,A767insASV, S768dupSVD, V769insASV, D770insSVD, D770insNPG, H773insNPH,N771del insGY, N771del insFH and N771dupNPH. In certain aspects, afavorable response to poziotinib inhibitor alone or in combination withan anti-cancer therapy comprises reduction in tumor size or burden,blocking of tumor growth, reduction in tumor-associated pain, reductionin cancer associated pathology, reduction in cancer associated symptoms,cancer non-progression, increased disease free interval, increased timeto progression, induction of remission, reduction of metastasis, orincreased patient survival. In further aspects, the patient predicted tohave a favorable response is administered poziotinib alone or incombination with a second anti-cancer therapy.

A further embodiment provides a method of treating cancer in a patientcomprising administering an effective amount of poziotinib or afatinibto the subject, wherein the subject has been determined to have one ormore HER2 exon 20 mutations selected from the group consisting ofA775insV G776C, A775insYVMA, G776C V777insC, G776del insVV, G776delinsVC, and P780insGSP. In some aspects, the one or more HER2 exon 20mutations further comprise one or more point mutations, insertions,and/or deletions of 3-18 nucleotides between amino acids 770-785. Insome aspects, the one or more HER2 exon 20 mutations are at residueA775, G776, S779, and/or P780. In particular aspects, the subject ishuman.

In some aspects, the method further comprises administering an mTORinhibitor. In certain aspects, the mTOR inhibitor is rapamycin,temsirolimus, everolimus, ridaforolimus or MLN4924. In particularaspects, the mTOR inhibitor is everolimus.

In certain aspects, the poziotinib or afatinib and/or mTOR inhibitor areadministered intravenously, subcutaneously, intraosseously, orally,transdermally, in sustained release, in controlled release, in delayedrelease, as a suppository, or sublingually. In some aspects, the patientwas determined to have a HER2 exon 20 mutation by analyzing a genomicsample from the patient. In certain aspects, the genomic sample isisolated from saliva, blood, urine, normal tissue, or tumor tissue. Insome aspects, the presence of an HER2 exon 20 mutation is determined bynucleic acid sequencing or PCR analyses.

In additional aspects, the method further comprises administering anadditional anti-cancer therapy. In some aspects, the anti-cancer therapyis chemotherapy, radiotherapy, gene therapy, surgery, hormonal therapy,anti-angiogenic therapy or immunotherapy.

In some aspects, the cancer is oral cancer, oropharyngeal cancer,nasopharyngeal cancer, respiratory cancer, urogenital cancer,gastrointestinal cancer, central or peripheral nervous system tissuecancer, an endocrine or neuroendocrine cancer or hematopoietic cancer,glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma,brain cancer, oropharyngeal cancer, nasopharyngeal cancer, renal cancer,biliary cancer, pheochromocytoma, pancreatic islet cell cancer,Li-Fraumeni tumors, thyroid cancer, parathyroid cancer, pituitarytumors, adrenal gland tumors, osteogenic sarcoma tumors, multipleneuroendocrine type I and type II tumors, breast cancer, lung cancer,head and neck cancer, prostate cancer, esophageal cancer, trachealcancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer,ovarian cancer, uterine cancer, cervical cancer, testicular cancer,colon cancer, rectal cancer or skin cancer. In certain aspects, thecancer is non-small cell lung cancer.

In another embodiment, there is provided a pharmaceutical compositioncomprising poziotinib or afatinib for a patient determined to have oneor more HER2 exon 20 mutations selected from the group consisting ofA775insV G776C, A775insYVMA, G776C V777insC, G776del insVV, G776delinsVC, and P780insGSP. In some aspects, the HER2 exon 20 mutationfurther comprises one or more point mutations, insertions, and/ordeletions of 3-18 nucleotides between amino acids 770-785. In someaspects, the HER2 exon 20 mutation is at residue A775, G776, S779,and/or P780. In some aspects, the patient is being treated with ananti-cancer therapy.

In yet another embodiment, there is provided a method of predicting aresponse to poziotinib or afatinib alone or in combination with ananti-cancer therapy in a patient having a cancer comprising detecting anHER2 exon 20 mutation (e.g., HER2 exon 20 insertion mutation) selectedfrom the group consisting of A775insV G776C, A775insYVMA, G776CV777insC, G776del insVV, G776del insVC, and P780insGSP in a genomicsample obtained from said patient, wherein if the sample is positive forthe presence of the HER2 exon 20 mutation, then the patient is predictedto have a favorable response to the poziotinib or afatinib alone or incombination with an anti-cancer therapy. In some aspects, the HER2 exon20 mutation further comprises one or more point mutations, insertions,and/or deletions of 3-18 nucleotides between amino acids 770-785. Incertain aspects, the HER2 exon 20 mutation is at residue A775, G776,S779, and/or P780.

In some aspects, the genomic sample is isolated from saliva, blood,urine, normal tissue, or tumor tissue. In certain aspects, the presenceof a HER2 exon 20 mutation is determined by nucleic acid sequencing orPCR analyses. In particular aspects, the anti-cancer therapy is an mTORinhibitor. In some aspects, a favorable response to poziotinib orafatinib inhibitor alone or in combination with an anti-cancer therapycomprises reduction in tumor size or burden, blocking of tumor growth,reduction in tumor-associated pain, reduction in cancer associatedpathology, reduction in cancer associated symptoms, cancernon-progression, increased disease free interval, increased time toprogression, induction of remission, reduction of metastasis, orincreased patient survival. In further aspects, the patient predicted tohave a favorable response is administered poziotinib alone or incombination with a second anti-cancer therapy.

Also provided herein is a composition comprising nucleic acids isolatedfrom human cancer cells; and a primer pair that can amplify at least afirst portion of exon 20 of a human EGFR or HER2 coding sequence. Insome aspects, the composition further comprises a labeled probe moleculethat can specifically hybridize to the first portion of exon 20 of thehuman EGFR or HER coding sequence when there is a mutation in thesequence. In certain aspects, the composition further comprises athermostable DNA polymerase. In some aspects, the composition furthercomprises dNTPS. In some aspects, the labeled probe hybridizes to thefirst portion of exon 20 of the human EGFR coding sequence when there isa mutation selected from the group consisting of A763insFQEA,A767insASV, S768dupSVD, V769insASV, D770insSVD, D770insNPG, H773insNPH,N771del insGY, N771del insFH, and N771dupNPH. In certain aspects, thelabeled probe hybridizes to the first portion of exon 20 of the humanHER2 coding sequence when there is a mutation selected from the groupconsisting of A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776CV777insC, G776del insVV, G776del insVC, and P780insGSP.

In another embodiment, there is provided an isolated nucleic acidencoding a mutant EGFR protein, wherein said mutant protein differs fromwild-type human EGFR by one or more EGFR exon 20 mutations comprising apoint mutation, insertion, and/or deletion of 3-18 nucleotides betweenamino acids 763-778. In some aspects, the one or more EGFR exon 20mutations are at one or more residues selected from the group consistingof A763, A767, 5768, V769, D770, N771, P772, and H773. In certainaspects, the one or more exon 20 mutations are selected from the groupconsisting of A763insFQEA, A767insASV, S768dupSVD, V769insASV,D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, andN771dupNPH. In specific aspects, the nucleic acid comprises the sequenceof SEQ ID NO:8, 9, 10, 11, or 12.

In yet another embodiment, there is provided an isolated nucleic acidencoding a mutant HER2 protein, wherein said mutant protein differs fromwild-type human HER2 by one or more HER2 exon 20 mutations comprisingone or more point mutations, insertions, and/or deletions of 3-18nucleotides between amino acids 770-785. In some aspects, the one ormore HER2 exon 20 mutations are at residue A775, G776, S779, and/orP780. In certain aspects, the one or more HER2 exon 20 mutationsselected from the group consisting of A775insV G776C, A775insYVMA,G776V, G776C V777insV, G776C V777insC, G776del insVV, G776del insVC, andP780insGSP. In specific aspects, the nucleic acid comprises the sequenceof SEQ ID NO:14, 15, 16, 17, or 18.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-1J: Exon 20 insertion mutations induce de novo resistance tocovalent and non-covalent TKIs. (FIG. 1A) Progression free survival(PFS) of patients with classical and exon 20 EGFR mutations demonstratesresistance to first line therapy. Patients with exon 20 insertions havedecreased percent survival. (FIG. 1B) Schematic of EGFR and HER2 exon 20insertion mutations generated in stable Ba/F3 model. Dose responsecurves of cell viability of Ba/F3 cell lines expressing EGFR (FIGS.1C-E) and (FIGS. 1F-H) HER2 exon 20 insertion mutations treated with1st, 2nd, and 3rd generation TKIs for 72 hours. (FIGS. 1C-H) Themean±SEM of 6 cell lines is plotted for each concentration (n=3). (FIG.1I) 3-D modeling of EGFR D770insNPG and T790M. Shifts of the P-loop andthe α-c-helix into the binding pocket result in steric hindrance,pushing AZD9291 out of the binding pocket. (FIG. 1J) 3-D modeling ofHER2 A775insYVMA and WT. Overall shifts of the P-loop and the α-c-helixinto the binding pocket result in an overall reduction in the size ofthe binding pocket.

FIGS. 2A-2G: Poziotinib potently inhibits EGFR and HER2 exon 20insertion mutations. Dose response curves of cell viability of Ba/F3cell lines expressing EGFR (FIG. 2A) and HER2 (FIG. 2B) exon 20insertion mutations treated with poziotinib for 72 hours. The mean±SEMof each individual cell line is plotted for each concentration (n=3).(FIG. 2C) Western blotting confirms inhibition of p-EGFR and p-HER2 inBa/F3 cell lines after 2 hours of poziotinib treatment (n=2). (FIG. 2D)Correlation of Ba/F3 EGFR exon 20 insertion location with amino acidlocation (n=2). Pearson correlation and p-value were determined usingGraphPad Prism. (FIG. 2E) Dose response curves of cell viability ofpatient derived cell line CUTO14 expressing EGFR A767dupASV and (FIG.2F) YUL0019 expressing EGFR N771del insFH treated with poziotinib orafatinib for 72 hours (n=3). (FIG. 2F) IC50 values of EGFR mutant Ba/F3cells normalized to the IC50 values of Ba/F3 EGFR T790M cell line afterincubation with afatinib, osimertinib, rociletinib, or poziotinib for 72hours (n=3). (FIG. 2G) Bars are representative of mean±SEM. Valuesgreater than 1 are indicative of less potent inhibition compared toT790M, whereas values less than one indicate more potent inhibition ofexon 20 insertions compared to T790M.

FIGS. 3A-3H: Poziotinib reduces tumor burden in EGFR and HER2 exon 20insertion mutation mouse models. EGFR D770insNPG (FIG. 3A) or HER2A775insYVMA (FIG. 3B) mice were treated daily with vehicle (EGFR n=5 andHER2 n=4), 20 mg/kg of afatinib (EGFR n=4), or 10 mg/kg of poziotinib(EGFR n=5 and HER2 n=6) for 4 weeks. Waterfall plots of tumor volumechange as measured by MRI demonstrate 85% and 60% tumor inhibition withpoziotinib at 4 weeks in EGFR and HER2 GEMMs, respectively. (FIGS. 3A-B)Two-sided student's t-test was used to calculate p-value. RepresentativeMRI images of EGFR (FIG. 3C) and HER2 (FIG. 3D) GEMM before and after 4weeks poziotinib treatment demonstrate robust tumor regression. Plots oftumor volume of EGFR D770insNPG (FIG. 3E) (n=4) and HER2 A775insYVMA(FIG. 3F) (n=6) treated with 10 mg/kg of poziotinib 5 days/week for 12weeks, exhibits mice continue to respond to poziotinib treatment. (FIG.3G) YUL-0019 (EGFR N771delinsFH) cells treated with afatinib orpoziotinib. The cells treated with 10 mg/kg poziotinib had the lowesttumor volume and with 5 mg/kg had the 2^(nd) to lowest tumor volume.(FIG. 3H) EGFR H773insNPH PDX mice were treated with vehicle control(n=6), 5 mg/kg (n=6) or 10 mg/kg (n=3) of poziotinib. The mice treatedwith poziotinib had decreased tumor volume. Waterfall plots demonstratethat tumor burden was reduced by >85% in all poziotinib treated mice,and in 8 out of 9 poziotinib treated mice, xenografts were completelyreduced to a residual bolus. One-way ANOVA analysis was used incombination with Tukey's test to determine statistical significance,***, p<0.0001.

FIGS. 4A-4C: EGFR and HiER2 exon 20 insertion mutations are activatingmutations. (FIG. 4A) Waterfall plots of individual patients with EGFRexon 20 insertions displays de novo resistance to erlotinib, geftinib,or afatinib. Patient mutations are listed below each representative bar.(FIG. 4B) Stable Ba/F3 cell lines expressing EGFR exon 20 insertionmutations are viable in IL-3 independent conditions, unlike Ba/F3 emptyvector expressing cells or EGFR WT expressing Ba/F3 cells, indicatingthat EGFR exon 20 insertions are activating mutations. (FIG. 4C) IL-3independent growth of 11 stable Ba/F3 cell lines expressing differentHER2 mutations displays that the majority of HER2 activating mutationsare within exon 20 of HER2. With the exception of L755P, all activatingmutations were HER2 exon 20 insertion mutations. (FIGS. 4B-C) Cellviability was determined by the Cell Titer Glo assay. The mean±SEM isplotted for each cell line (n=3).

FIG. 5 : Dose response curves of cell viability of individual Ba/F3 celllines expressing EGFR exon 20 insertion mutations treated with 1st, 2nd,and 3rd generation TKIs for 72 hours. The mean±SEM is plotted for eachconcentration (n=3).

FIG. 6 : Dose response curves of cell viability of individual Ba/F3 celllines expressing HER2 exon 20 insertion mutations treated with 1st, 2nd,and 3rd generation TKIs for 72 hours. The mean±SEM is plotted for eachconcentration (n=3).

FIGS. 7A-7D: EGFR and HER2 exon 20 insertions mutations after residueA763 are resistant to 1st and 3rd generation TKIs. Ba/F3 cells with EGFRexon 20 insertions were serum starved for 1 hour then treated withindicated doses of (FIG. 7A) erlotinib or (FIG. 7C) osimertinib for 2hours (N=2). p-EGFR and p-HER2 levels after (FIG. 7B) erlotinibtreatment and (FIG. 7D) osimertinib treatment were quantified usingPhotoshop. Values were plotted in Graphpad Prism and bars arerepresentative of mean±SEM. (N=2) p<0.05 (*), p<0.01 (**) or p<0.001(***).

FIGS. 8A-8E: EGFR and HER2 exon 20 insertions mutations are sensitive topoziotinib in vitro. (FIG. 8A) Western blots of p-EGFR and p-HER2 after2 hours of poziotinib treatment in indicated Ba/F3 cell lines werequantified using Photoshop. Values were plotted in Graphpad Prism andbars are representative of mean±SEM. (N=2) (FIG. 8B) Western blot ofCUTO-14 patient derived cell line after 3 hours of indicated doses ofafatinib or poziotinib (N=3). (FIG. 8C) Quantification of p-EGFR fromwestern blots after 3 hours of indicated doses of afatinib or poziotinibin CUTO-14 cell line. Poziotinib treatment resulted in decreased p-EGFR.(FIG. 8D) Linear regression plot of IC50 values vs. relative expressionof Ba/F3 cell lines demonstrated that there was no correlation betweenexpression and sensitivity to poziotinib (n=2). (FIG. 8E) Linearregression plot of IC50 values vs. the location of the mutation withinthe HER2 receptor demonstrated that there was no correlation betweenlocation and sensitivity to poziotinib in HER2 mutant Ba/F3 cell lines(n=2). Pearson correlations and p-values were calculated using Graphpadprism. p<0.05 (*), p<0.01 (**) or p<0.001 (***).

FIG. 9 : C797S and EMT are two distinct mechanisms of poziotinibresistance in vitro. Dose response curves of cell viability of EGFRmutant Ba/F3 cell lines treated with poziotinib for 72 hours. Themean±SEM is plotted for each concentration (n=3).

FIG. 10 : Dose response curves of cell viability of MCF10A HER2 G776delinsVC cell line treated with indicated TKIs.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Although the majority of activating mutations of epidermal growth factorreceptor (EGFR) mutant non-small cell lung cancers (NSCLCs) aresensitive to available EGFR tyrosine kinase inhibitor (TKIs), a subsetwith alterations in exon 20 of EGFR and HER2 are intrinsicallyresistant. The present studies utilized in silico, in vitro, and in vivotesting to model structural alterations induced by these exon 20mutations and identify effective inhibitors. 3-D modeling revealedsignificant alterations restricting the size of the drug binding pocket,imposing the binding of large, rigid inhibitors. It was found thatpoziotinib, due to its small size and flexibility, was able tocircumvent these steric changes, and is a potent and relativelyselective inhibitor of the EGFR or HER2 exon 20 mutant proteins.Poziotinib also has potent activity in mutant exon 20 EGFR or HER2 NSCLCpatient-derived xenograft (PDX) models and genetically engineered mousemodels. Thus, these data identify poziotinib as a potent, clinicallyactive inhibitor of EGFR/HER2 exon 20 mutations, and illuminate themolecular features of kinase inhibitors that may circumvent stericchanges induced by these insertions.

Accordingly, certain embodiments of the present disclosure providemethods for treating cancer patients with EGFR and/or HER2 exon 20mutations, such as exon 20 insertions. In particular, the presentmethods comprise the administration of poziotinib (also known asHM781-36B) or afatinib to patients identified to have EGFR and/or HERexon 20 insertion mutations. The size and flexibility of poziotinibovercomes steric hindrance, inhibiting EGFR and HER2 exon 20 mutants atlow nanomolar concentrations. Thus, poziotinib or afatinib as well asstructurally similar inhibitors are potent EGFR or HER2 inhibitors thatcan be used to target both EFGR and HER2 exon 20 insertions which areresistant to irreversible 2^(nd) and 3^(rd) generations TKIs.

I. Definitions

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising,” the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease. For example, a treatment may includeadministration of an effective amount of poziotinib or afatinib.

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate. Non-limiting examplesof human patients are adults, juveniles, infants and fetuses.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult. “Effective amount,” “therapeutically effective amount” or“pharmaceutically effective amount” when used in the context of treatinga patient or subject with a compound means that amount of the compoundwhich, when administered to a subject or patient for treating orpreventing a disease, is an amount sufficient to effect such treatmentor prevention of the disease.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the maximum response obtained. This quantitative measureindicates how much of a particular drug or other substance (inhibitor)is needed to inhibit a given biological, biochemical or chemical process(or component of a process, i.e. an enzyme, cell, cell receptor ormicroorganism) by half.

An “anti-cancer” agent is capable of negatively affecting a cancercell/tumor in a subject, for example, by promoting killing of cancercells, inducing apoptosis in cancer cells, reducing the growth rate ofcancer cells, reducing the incidence or number of metastases, reducingtumor size, inhibiting tumor growth, reducing the blood supply to atumor or cancer cells, promoting an immune response against cancer cellsor a tumor, preventing or inhibiting the progression of cancer, orincreasing the lifespan of a subject with cancer.

The term “insertion(s)” or “insertion mutation(s)” refers to theaddition of one or more nucleotide base pairs into a DNA sequence. Forexample, an insertion mutation of exon 20 of EGFR can occur betweenamino acids 767 to 774, of about 2-21 base pairs. In another example,HER2 exon 20 insertion mutation comprises one or more insertions of 3-18nucleotides between amino acids 770-785. Exemplary EGFR and HER exon 20insertion mutations are depicted in FIG. 1 of the present disclosure.

“Hybridize” or “hybridization” refers to the binding between nucleicacids. The conditions for hybridization can be varied according to thesequence homology of the nucleic acids to be bound. Thus, if thesequence homology between the subject nucleic acids is high, stringentconditions are used. If the sequence homology is low, mild conditionsare used. When the hybridization conditions are stringent, thehybridization specificity increases, and this increase of thehybridization specificity leads to a decrease in the yield ofnon-specific hybridization products. However, under mild hybridizationconditions, the hybridization specificity decreases, and this decreasein the hybridization specificity leads to an increase in the yield ofnon-specific hybridization products.

A “probe” or “probes” refers to a polynucleotide that is at least eight(8) nucleotides in length and which forms a hybrid structure with atarget sequence, due to complementarity of at least one sequence in theprobe with a sequence in the target region. The polynucleotide can becomposed of DNA and/or RNA. Probes in certain embodiments, aredetectably labeled. Probes can vary significantly in size. Generally,probes are, for example, at least 8 to 15 nucleotides in length. Otherprobes are, for example, at least 20, 30 or 40 nucleotides long. Stillother probes are somewhat longer, being at least, for example, 50, 60,70, 80, or 90 nucleotides long. Probes can be of any specific lengththat falls within the foregoing ranges as well. Preferably, the probedoes not contain a sequence complementary to the sequence(s) used toprime for a target sequence during the polymerase chain reaction.

“Oligonucleotide” or “polynucleotide” refers to a polymer of asingle-stranded or double-stranded deoxyribonucleotide orribonucleotide, which may be unmodified RNA or DNA or modified RNA orDNA.

A “modified ribonucleotide” or deoxyribonucleotide refer to moleculesthat can be used in place of naturally occurring bases in nucleic acidand includes, but is not limited to, modified purines and pyrimidines,minor bases, convertible nucleosides, structural analogs of purines andpyrimidines, labeled, derivatized and modified nucleosides andnucleotides, conjugated nucleosides and nucleotides, sequence modifiers,terminus modifiers, spacer modifiers, and nucleotides with backbonemodifications, including, but not limited to, ribose-modifiednucleotides, phosphoramidates, phosphorothioates, phosphonamidites,methyl phosphonates, methyl phosphoramidites, methyl phosphonamidites,5′-β-cyanoethyl phosphoramidites, methylenephosphonates,phosphorodithioates, peptide nucleic acids, achiral and neutralinternucleotidic linkages.

A “variant” refers to a polynucleotide or polypeptide that differsrelative to a wild-type or the most prevalent form in a population ofindividuals by the exchange, deletion, or insertion of one or morenucleotides or amino acids, respectively. The number of nucleotides oramino acids exchanged, deleted, or inserted can be 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more such as 25, 30,35, 40, 45 or 50.

A “primer” or “primer sequence” refers to an oligonucleotide thathybridizes to a target nucleic acid sequence (for example, a DNAtemplate to be amplified) to prime a nucleic acid synthesis reaction.The primer may be a DNA oligonucleotide, a RNA oligonucleotide, or achimeric sequence. The primer may contain natural, synthetic, ormodified nucleotides. Both the upper and lower limits of the length ofthe primer are empirically determined. The lower limit on primer lengthis the minimum length that is required to form a stable duplex uponhybridization with the target nucleic acid under nucleic acidamplification reaction conditions. Very short primers (usually less than3-4 nucleotides long) do not form thermodynamically stable duplexes withtarget nucleic acid under such hybridization conditions. The upper limitis often determined by the possibility of having a duplex formation in aregion other than the pre-determined nucleic acid sequence in the targetnucleic acid. Generally, suitable primer lengths are in the range ofabout 10 to about 40 nucleotides long. In certain embodiments, forexample, a primer can be 10-40, 15-30, or 10-20 nucleotides long. Aprimer is capable of acting as a point of initiation of synthesis on apolynucleotide sequence when placed under appropriate conditions.

“Detection,” “detectable” and grammatical equivalents thereof refers toways of determining the presence and/or quantity and/or identity of atarget nucleic acid sequence. In some embodiments, detection occursamplifying the target nucleic acid sequence. In other embodiments,sequencing of the target nucleic acid can be characterized as“detecting” the target nucleic acid. A label attached to the probe caninclude any of a variety of different labels known in the art that canbe detected by, for example, chemical or physical means. Labels that canbe attached to probes may include, for example, fluorescent andluminescence materials.

“Amplifying,” “amplification,” and grammatical equivalents thereofrefers to any method by which at least a part of a target nucleic acidsequence is reproduced in a template-dependent manner, including withoutlimitation, a broad range of techniques for amplifying nucleic acidsequences, either linearly or exponentially. Exemplary means forperforming an amplifying step include ligase chain reaction (LCR),ligase detection reaction (LDR), ligation followed by Q-replicaseamplification, PCR, primer extension, strand displacement amplification(SDA), hyperbranched strand displacement amplification, multipledisplacement amplification (MDA), nucleic acid strand-basedamplification (NASBA), two-step multiplexed amplifications, rollingcircle amplification (RCA), recombinase-polymerase amplification (RPA)(TwistDx, Cambridg, UK), and self-sustained sequence replication (3SR),including multiplex versions or combinations thereof, for example butnot limited to, OLA/PCR, PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR,LCR/PCR, PCR/LCR (also known as combined chain reaction-CCR), and thelike. Descriptions of such techniques can be found in, among otherplaces, Sambrook et al. Molecular Cloning, 3^(rd) Edition).

“EGFR” or “Epidermal growth factor receptor” or “EGFR” refers to atyrosine kinase cell surface receptor and is encoded by one of fouralternative transcripts appearing as GenBank accession NM_005228.3,NM_201282.1, NM_201283.1 and NM_201284.1. Variants of EGFR include aninsertion in exon 20.

“HER2” or “ERBB2” is a member of the EGFR/ErbB family and appears asGenBank accession NM_004448.2. Variants of HER2 include an insertion inexon 20.

As generally used herein “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent invention which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity.Non-limiting examples of such salts include acid addition salts formedwith inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, and phosphoric acid; or with organic acidssuch as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, and trimethylacetic acid. Pharmaceuticallyacceptable salts also include base addition salts which may be formedwhen acidic protons present are capable of reacting with inorganic ororganic bases. Acceptable inorganic bases include sodium hydroxide,sodium carbonate, potassium hydroxide, aluminum hydroxide and calciumhydroxide. Non-limiting examples of acceptable organic bases includeethanolamine, diethanolamine, triethanolamine, tromethamine, andN-methylglucamine. It should be recognized that the particular anion orcation forming a part of any salt of this invention is not critical, solong as the salt, as a whole, is pharmacologically acceptable.Additional examples of pharmaceutically acceptable salts and theirmethods of preparation and use are presented in Handbook ofPharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermutheds., Verlag Helvetica Chimica Acta, 2002).

II. EGFR and HER2 Exon 20 Mutations

Certain embodiments of the present disclosure concern determining if asubject has one or more EGFR and/or HER2 exon 20 mutations, such as aninsertion mutations, particularly one or more insertion mutations asdepicted in FIG. 1 . The subject may have 2, 3, 4, or more EGFR exon 20mutations and/or HER2 exon 20 mutations. Mutation detection methods areknown the art including PCR analyses and nucleic acid sequencing as wellas FISH and CGH. In particular aspects, the exon 20 mutations aredetected by DNA sequencing, such as from a tumor or circulating free DNAfrom plasma.

The EGFR exon 20 mutation(s) may comprise one or more point mutations,insertions, and/or deletions of 3-18 nucleotides between amino acids763-778. The one or more EGFR exon 20 mutations may be located at one ormore residues selected from the group consisting of A763, A767, S768,V769, D770, N771, P772, and H773.

EGFR exon 20 insertions may include H773_V774insH, A767_v769ASV,N771_P772insH, D770_N771insG, H779_V774insH, N771delinsHH,S768_D770dupDVD, A767_V769dupASV, A767_V769dupASV, P772_H773dup,N771_H773dupNPH, S768_D770dupSVD, N771delinsGY, S768_D770delinsSVD,D770_D770delinsGY, A767_V769dupASV, and/or H773dup. In particularaspects, the exon 20 mutations are A763insFQEA, A767insASV, S768dupSVD,V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771delinsFH and/or N771dupNPH.

In some aspects, the subject may have or develop a mutation at EGFRresidue C797 which may result in resistance to the TKI, such aspoziotinib. Thus, in certain aspects, the subject is determined to nothave a mutation at EGFR C797.

The HER2 exon 20 mutation may comprise one or more point mutations,insertions, and/or deletions of 3-18 nucleotides between amino acids770-785. The one or more HER2 exon 20 mutations may be at residue A775,G776, S779, and/or P780. The one or more HER2 exon 20 mutations may beA775insV G776C, A775insYVMA, G776V, G776C V777insV, G776C V777insC,G776del insVV, G776del insVC, and/or P780insGSP.

The patient sample can be any bodily tissue or fluid that includesnucleic acids from the lung cancer in the subject. In certainembodiments, the sample will be a blood sample comprising circulatingtumor cells or cell free DNA. In other embodiments, the sample can be atissue, such as a lung tissue. The lung tissue can be from a tumortissue and may be fresh frozen or formalin-fixed, paraffin-embedded(FFPE). In certain embodiments, a lung tumor FFPE sample is obtained.

Samples that are suitable for use in the methods described hereincontain genetic material, e.g., genomic DNA (gDNA). Genomic DNA istypically extracted from biological samples such as blood or mucosalscrapings of the lining of the mouth, but can be extracted from otherbiological samples including urine, tumor, or expectorant. The sampleitself will typically include nucleated cells (e.g., blood or buccalcells) or tissue removed from the subject including normal or tumortissue. Methods and reagents are known in the art for obtaining,processing, and analyzing samples. In some embodiments, the sample isobtained with the assistance of a health care provider, e.g., to drawblood. In some embodiments, the sample is obtained without theassistance of a health care provider, e.g., where the sample is obtainednon-invasively, such as a sample comprising buccal cells that isobtained using a buccal swab or brush, or a mouthwash sample.

In some cases, a biological sample may be processed for DNA isolation.For example, DNA in a cell or tissue sample can be separated from othercomponents of the sample. Cells can be harvested from a biologicalsample using standard techniques known in the art. For example, cellscan be harvested by centrifuging a cell sample and resuspending thepelleted cells. The cells can be resuspended in a buffered solution suchas phosphate-buffered saline (PBS). After centrifuging the cellsuspension to obtain a cell pellet, the cells can be lysed to extractDNA, e.g., gDNA. See, e.g., Ausubel et al. (2003). The sample can beconcentrated and/or purified to isolate DNA. All samples obtained from asubject, including those subjected to any sort of further processing,are considered to be obtained from the subject. Routine methods can beused to extract genomic DNA from a biological sample, including, forexample, phenol extraction. Alternatively, genomic DNA can be extractedwith kits such as the QIAamp® Tissue Kit (Qiagen, Chatsworth, Calif.)and the Wizard® Genomic DNA purification kit (Promega). Non-limitingexamples of sources of samples include urine, blood, and tissue.

The presence or absence of EGFR or HER2 exon 20 mutations, such as anexon 20 insertion mutation, as described herein can be determined usingmethods known in the art. For example, gel electrophoresis, capillaryelectrophoresis, size exclusion chromatography, sequencing, and/orarrays can be used to detect the presence or absence of insertionmutations. Amplification of nucleic acids, where desirable, can beaccomplished using methods known in the art, e.g., PCR. In one example,a sample (e.g., a sample comprising genomic DNA), is obtained from asubject. The DNA in the sample is then examined to determine theidentity of an insertion mutation as described herein. An insertionmutation can be detected by any method described herein, e.g., bysequencing or by hybridization of the gene in the genomic DNA, RNA, orcDNA to a nucleic acid probe, e.g., a DNA probe (which includes cDNA andoligonucleotide probes) or an RNA probe. The nucleic acid probe can bedesigned to specifically or preferentially hybridize with a particularvariant.

A set of probes typically refers to a set of primers, usually primerpairs, and/or detectably-labeled probes that are used to detect thetarget genetic variations (e.g., EGFR and/or HER2 exon 20 mutations)used in the actionable treatment recommendations of the presentdisclosure. The primer pairs are used in an amplification reaction todefine an amplicon that spans a region for a target genetic variationfor each of the aforementioned genes. The set of amplicons are detectedby a set of matched probes. In an exemplary embodiment, the presentmethods may use TaqMan™ (Roche Molecular Systems, Pleasanton, Calif.)assays that are used to detect a set of target genetic variations, suchas EGFR and/or HER2 exon 20 mutations. In one embodiment, the set ofprobes are a set of primers used to generate amplicons that are detectedby a nucleic acid sequencing reaction, such as a next generationsequencing reaction. In these embodiments, for example, AmpliSEQ™ (LifeTechnologies/Ion Torrent, Carlsbad, Calif.) or TruSEQ™ (Illumina, SanDiego, Calif.) technology can be employed.

Analysis of nucleic acid markers can be performed using techniques knownin the art including, without limitation, sequence analysis, andelectrophoretic analysis. Non-limiting examples of sequence analysisinclude Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNAsequencing, thermal cycle sequencing (Sears et al., 1992), solid-phasesequencing (Zimmerman et al., 1992), sequencing with mass spectrometrysuch as matrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF/MS; Fu et al., 1998), and sequencing byhybridization (Chee et al., 1996; Drmanac et al., 1993; Drmanac et al.,1998). Non-limiting examples of electrophoretic analysis include slabgel electrophoresis such as agarose or polyacrylamide gelelectrophoresis, capillary electrophoresis, and denaturing gradient gelelectrophoresis. Additionally, next generation sequencing methods can beperformed using commercially available kits and instruments fromcompanies such as the Life Technologies/Ion Torrent PGM or Proton, theIllumina HiSEQ or MiSEQ, and the Roche/454 next generation sequencingsystem.

Other methods of nucleic acid analysis can include direct manualsequencing (Church and Gilbert, 1988; Sanger et al., 1977; U.S. Pat. No.5,288,644); automated fluorescent sequencing; single-strandedconformation polymorphism assays (SSCP) (Schafer et al., 1995); clampeddenaturing gel electrophoresis (CDGE); two-dimensional gelelectrophoresis (2DGE or TDGE); conformational sensitive gelelectrophoresis (CSGE); denaturing gradient gel electrophoresis (DGGE)(Sheffield et al., 1989); denaturing high performance liquidchromatography (DHPLC, Underhill et al., 1997); infrared matrix-assistedlaser desorption/ionization (IR-MALDI) mass spectrometry (WO 99/57318);mobility shift analysis (Orita et al., 1989); restriction enzymeanalysis (Flavell et al., 1978; Geever et al., 1981); quantitativereal-time PCR (Raca et al., 2004); heteroduplex analysis; chemicalmismatch cleavage (CMC) (Cotton et al., 1985); RNase protection assays(Myers et al., 1985); use of polypeptides that recognize nucleotidemismatches, e.g., E. coli mutS protein; allele-specific PCR, andcombinations of such methods. See, e.g., U.S. Patent Publication No.2004/0014095, which is incorporated herein by reference in its entirety.

In one example, a method of identifying an EGFR and/or HER2 mutation ina sample comprises contacting a nucleic acid from said sample with anucleic acid probe that is capable of specifically hybridizing tonucleic acid encoding a mutated EGFR or HER2 protein, or fragmentthereof incorporating a mutation, and detecting said hybridization. In aparticular embodiment, said probe is detectably labeled such as with aradioisotope (³H, ³²P, or ³³P), a fluorescent agent (rhodamine, orfluorescein) or a chromogenic agent. In a particular embodiment, theprobe is an antisense oligomer, for example PNA,morpholino-phosphoramidates, LNA or 2′-alkoxyalkoxy. The probe may befrom about 8 nucleotides to about 100 nucleotides, or about 10 to about75, or about 15 to about 50, or about 20 to about 30. In another aspect,said probes of the present disclosure are provided in a kit foridentifying EGFR or HER2 mutations in a sample, said kit comprising anoligonucleotide that specifically hybridizes to or adjacent to a site ofmutation in the EGFR or HER2 gene. The kit may further compriseinstructions for treating patients having tumors that contain EGFR orHER2 insertion mutations with poziotinib or afatinib based on the resultof a hybridization test using the kit.

In another aspect, a method for detecting an exon 20 mutation in asample comprises amplifying from said sample nucleic acids correspondingto exon 20 of said EGFR gene or HER2, or a fragment thereof suspected ofcontaining a mutation, and comparing the electrophoretic mobility of theamplified nucleic acid to the electrophoretic mobility of correspondingwild-type EGFR or HER2 gene or fragment thereof. A difference in themobility indicates the presence of a mutation in the amplified nucleicacid sequence. Electrophoretic mobility may be determined onpolyacrylamide gel.

Alternatively, nucleic acids may be analyzed for detection of mutationsusing Enzymatic Mutation Detection (EMD) (Del Tito et al., 1998). EMDuses the bacteriophage resolvase T4 endonuclease VII, which scans alongdouble-stranded DNA until it detects and cleaves structural distortionscaused by base pair mismatches resulting from point mutations,insertions and deletions. Detection of two short fragments formed byresolvase cleavage, for example by gel electrophoresis, indicates thepresence of a mutation. Benefits of the EMD method are a single protocolto identify point mutations, deletions, and insertions assayed directlyfrom PCR reactions eliminating the need for sample purification,shortening the hybridization time, and increasing the signal-to-noiseratio. Mixed samples containing up to a 20-fold excess of normal DNA andfragments up to 4 kb in size can been assayed. However, EMD scanningdoes not identify particular base changes that occur in mutationpositive samples requiring additional sequencing procedures to identityof the mutation if necessary. CEL I enzyme can be used similarly toresolvase T4 endonuclease VII as demonstrated in U.S. Pat. No.5,869,245.

III. Methods of Treatment

Further provided herein are methods for treating or delaying progressionof cancer in an individual comprising administering to the individual aneffective amount of poziotinib, afatinib, or a structurally similarinhibitor, to a subject determined to have an EGFR and/or HER2 exon 20mutations, such as an exon 20 insertion. The subject may have more thanone EGFR and/or HER exon 20 mutation.

Examples of cancers contemplated for treatment include lung cancer, headand neck cancer, breast cancer, pancreatic cancer, prostate cancer,renal cancer, bone cancer, testicular cancer, cervical cancer,gastrointestinal cancer, lymphomas, pre-neoplastic lesions in the lung,colon cancer, melanoma, and bladder cancer. In particular aspects, thecancer is non-small cell lung cancer.

In some embodiments, the subject is a mammal, e.g., a primate,preferably a higher primate, e.g., a human (e.g., a patient having, orat risk of having, a disorder described herein). In one embodiment, thesubject is in need of enhancing an immune response. In certainembodiments, the subject is, or is at risk of being, immunocompromised.For example, the subject is undergoing or has undergone achemotherapeutic treatment and/or radiation therapy. Alternatively, orin combination, the subject is, or is at risk of being,immunocompromised as a result of an infection.

Certain embodiments concern the administration of poziotinib (also knownas HM781-36B, HM781-36, and1-[4-[4-(3,4-dichloro-2-fluoroanilino)-7-methoxyquinazolin-6-yl]oxypiperidin-1-yl]prop-2-en-1-one)to a subject determined to have EGFR or HER2 exon 20 mutation, such asan exon 20 insertion. Poziotinib is a quinazoline-based pan-HERinhibitor that irreversibly blocks signaling through the HER family oftyrosine-kinase receptors including HER1, HER2, and HER4. Poziotinib orstructurally similar compounds (e.g., U.S. Pat. No. 8,188,102 and U.S.Patent Publication No. 20130071452; incorporated herein by reference)may be used in the present methods.

B. Pharmaceutical Compositions

Also provided herein are pharmaceutical compositions and formulationscomprising poziotinib or afatinib and a pharmaceutically acceptablecarrier for subjects determined to have an EGFR or HER2 exon 20mutation, such as an exon 20 insertion.

Pharmaceutical compositions and formulations as described herein can beprepared by mixing the active ingredients (such as an antibody or apolypeptide) having the desired degree of purity with one or moreoptional pharmaceutically acceptable carriers (Remington'sPharmaceutical Sciences 22^(nd) edition, 2012), in the form oflyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable carriers are generally nontoxic to recipients at the dosagesand concentrations employed, and include, but are not limited to:buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in U.S. Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

C. Combination Therapies

In certain embodiments, the compositions and methods of the presentembodiments involve poziotinib or afatinib in combination with at leastone additional therapy. The additional therapy may be radiation therapy,surgery (e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy,DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrowtransplantation, nanotherapy, monoclonal antibody therapy, or acombination of the foregoing. The additional therapy may be in the formof adjuvant or neoadjuvant therapy.

In some embodiments, the additional therapy is the administration ofsmall molecule enzymatic inhibitor or anti-metastatic agent. In someembodiments, the additional therapy is the administration of side-effectlimiting agents (e.g., agents intended to lessen the occurrence and/orseverity of side effects of treatment, such as anti-nausea agents,etc.). In some embodiments, the additional therapy is radiation therapy.In some embodiments, the additional therapy is surgery. In someembodiments, the additional therapy is a combination of radiationtherapy and surgery. In some embodiments, the additional therapy isgamma irradiation. In some embodiments, the additional therapy istherapy targeting PBK/AKT/mTOR pathway, HSP90 inhibitor, tubulininhibitor, apoptosis inhibitor, and/or chemopreventative agent. Theadditional therapy may be one or more of the chemotherapeutic agentsknown in the art.

The poziotinib or afatinib may be administered before, during, after, orin various combinations relative to an additional cancer therapy, suchas immune checkpoint therapy. The administrations may be in intervalsranging from concurrently to minutes to days to weeks. In embodimentswhere the poziotinib or afatinib is provided to a patient separatelyfrom an additional therapeutic agent, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the two compounds would still be able to exert anadvantageously combined effect on the patient. In such instances, it iscontemplated that one may provide a patient with the antibody therapyand the anti-cancer therapy within about 12 to 24 or 72 h of each otherand, more particularly, within about 6-12 h of each other. In somesituations it may be desirable to extend the time period for treatmentsignificantly where several days (2, 3, 4, 5, 6, or 7) to several weeks(1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

Various combinations may be employed. For the example below poziotinibor afatinib is “A” and an anti-cancer therapy is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of any compound or therapy of the present embodiments toa patient will follow general protocols for the administration of suchcompounds, taking into account the toxicity, if any, of the agents.Therefore, in some embodiments there is a step of monitoring toxicitythat is attributable to combination therapy.

1. Chemotherapy

A wide variety of chemotherapeutic agents may be used in accordance withthe present embodiments. The term “chemotherapy” refers to the use ofdrugs to treat cancer. A “chemotherapeutic agent” is used to connote acompound or composition that is administered in the treatment of cancer.These agents or drugs are categorized by their mode of activity within acell, for example, whether and at what stage they affect the cell cycle.Alternatively, an agent may be characterized based on its ability todirectly cross-link DNA, to intercalate into DNA, or to inducechromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents, such asthiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan,improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines, includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, and uracil mustard;nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; antibiotics, such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin gammalI andcalicheamicin omegaI1); dynemicin, including dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolicacid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; anti-metabolites, such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogues, such asdenopterin, pteropterin, and trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidineanalogs, such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;androgens, such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone; anti-adrenals, such as mitotane andtrilostane; folic acid replenisher, such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharidecomplex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g.,paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine;platinum coordination complexes, such as cisplatin, oxaliplatin, andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan(e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids, such as retinoic acid;capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien,navelbine, farnesyl-protein tansferase inhibitors, transplatinum, andpharmaceutically acceptable salts, acids, or derivatives of any of theabove.

2. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated, such as microwaves, proton beamirradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), andUV-irradiation. It is most likely that all of these factors affect abroad range of damage on DNA, on the precursors of DNA, on thereplication and repair of DNA, and on the assembly and maintenance ofchromosomes. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (3 to 4 wk), to single dosesof 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely,and depend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by the neoplastic cells.

3. Immunotherapy

The skilled artisan will understand that additional immunotherapies maybe used in combination or in conjunction with methods of theembodiments. In the context of cancer treatment, immunotherapeutics,generally, rely on the use of immune effector cells and molecules totarget and destroy cancer cells. Rituximab (RITUXAN®) is such anexample. The immune effector may be, for example, an antibody specificfor some marker on the surface of a tumor cell. The antibody alone mayserve as an effector of therapy or it may recruit other cells toactually affect cell killing. The antibody also may be conjugated to adrug or toxin (chemotherapeutic, radionuclide, ricin A chain, choleratoxin, pertussis toxin, etc.) and serve as a targeting agent.Alternatively, the effector may be a lymphocyte carrying a surfacemolecule that interacts, either directly or indirectly, with a tumorcell target. Various effector cells include cytotoxic T cells and NKcells

Antibody-drug conjugates have emerged as a breakthrough approach to thedevelopment of cancer therapeutics. Cancer is one of the leading causesof deaths in the world. Antibody-drug conjugates (ADCs) comprisemonoclonal antibodies (MAbs) that are covalently linked to cell-killingdrugs. This approach combines the high specificity of MAbs against theirantigen targets with highly potent cytotoxic drugs, resulting in “armed”MAbs that deliver the payload (drug) to tumor cells with enriched levelsof the antigen. Targeted delivery of the drug also minimizes itsexposure in normal tissues, resulting in decreased toxicity and improvedtherapeutic index. The approval of two ADC drugs, ADCETRIS® (brentuximabvedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013by FDA validated the approach. There are currently more than 30 ADC drugcandidates in various stages of clinical trials for cancer treatment(Leal et al., 2014). As antibody engineering and linker-payloadoptimization are becoming more and more mature, the discovery anddevelopment of new ADCs are increasingly dependent on the identificationand validation of new targets that are suitable to this approach and thegeneration of targeting MAbs. Two criteria for ADC targets areupregulated/high levels of expression in tumor cells and robustinternalization.

In one aspect of immunotherapy, the tumor cell must bear some markerthat is amenable to targeting, i.e., is not present on the majority ofother cells. Many tumor markers exist and any of these may be suitablefor targeting in the context of the present embodiments. Common tumormarkers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68,TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor,erb B, and p155. An alternative aspect of immunotherapy is to combineanticancer effects with immune stimulatory effects. Immune stimulatingmolecules also exist including: cytokines, such as IL-2, IL-4, IL-12,GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growthfactors, such as FLT3 ligand.

Examples of immunotherapies include immune adjuvants, e.g.,Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, andaromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui andHashimoto, 1998; Christodoulides et al., 1998); cytokine therapy, e.g.,interferons α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998;Davidson et al., 1998; Hellstrand et al., 1998); gene therapy, e.g.,TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca,1998; U.S. Pat. Nos. 5,830,880 and 5,846,945); and monoclonalantibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-p185(Hollander, 2012; Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311). Itis contemplated that one or more anti-cancer therapies may be employedwith the antibody therapies described herein.

In some embodiments, the immunotherapy may be an immune checkpointinhibitor. Immune checkpoints either turn up a signal (e.g.,co-stimulatory molecules) or turn down a signal. Inhibitory immunecheckpoints that may be targeted by immune checkpoint blockade includeadenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and Tlymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO),killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAGS),programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA). Inparticular, the immune checkpoint inhibitors target the PD-1 axis and/orCTLA-4.

The immune checkpoint inhibitors may be drugs such as small molecules,recombinant forms of ligand or receptors, or, in particular, areantibodies, such as human antibodies (e.g., International PatentPublication WO2015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012;both incorporated herein by reference). Known inhibitors of the immunecheckpoint proteins or analogs thereof may be used, in particularchimerized, humanized or human forms of antibodies may be used. As theskilled person will know, alternative and/or equivalent names may be inuse for certain antibodies mentioned in the present disclosure. Suchalternative and/or equivalent names are interchangeable in the contextof the present invention. For example it is known that lambrolizumab isalso known under the alternative and equivalent names MK-3475 andpembrolizumab.

In some embodiments, the PD-1 binding antagonist is a molecule thatinhibits the binding of PD-1 to its ligand binding partners. In aspecific aspect, the PD-1 ligand binding partners are PDL1 and/or PDL2.In another embodiment, a PDL1 binding antagonist is a molecule thatinhibits the binding of PDL1 to its binding partners. In a specificaspect, PDL1 binding partners are PD-1 and/or B7-1. In anotherembodiment, the PDL2 binding antagonist is a molecule that inhibits thebinding of PDL2 to its binding partners. In a specific aspect, a PDL2binding partner is PD-1. The antagonist may be an antibody, an antigenbinding fragment thereof, an immunoadhesin, a fusion protein, oroligopeptide. Exemplary antibodies are described in U.S. Pat. Nos.8,735,553, 8,354,509, and 8,008,449, all incorporated herein byreference. Other PD-1 axis antagonists for use in the methods providedherein are known in the art such as described in U.S. Patent PublicationNos. US20140294898, US2014022021, and US20110008369, all incorporatedherein by reference.

In some embodiments, the PD-1 binding antagonist is an anti-PD-1antibody (e.g., a human antibody, a humanized antibody, or a chimericantibody). In some embodiments, the anti-PD-1 antibody is selected fromthe group consisting of nivolumab, pembrolizumab, and CT-011. In someembodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., animmunoadhesin comprising an extracellular or PD-1 binding portion ofPDL1 or PDL2 fused to a constant region (e.g., an Fc region of animmunoglobulin sequence). In some embodiments, the PD-1 bindingantagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106,ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described inWO2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475,lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibodydescribed in WO2009/114335. CT-011, also known as hBAT or hBAT-1, is ananti-PD-1 antibody described in WO2009/101611. AMP-224, also known asB7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010/027827and WO2011/066342.

Another immune checkpoint that can be targeted in the methods providedherein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), alsoknown as CD152. The complete cDNA sequence of human CTLA-4 has theGenbank accession number L15006. CTLA-4 is found on the surface of Tcells and acts as an “off” switch when bound to CD80 or CD86 on thesurface of antigen-presenting cells. CTLA4 is a member of theimmunoglobulin superfamily that is expressed on the surface of Helper Tcells and transmits an inhibitory signal to T cells. CTLA4 is similar tothe T-cell co-stimulatory protein, CD28, and both molecules bind to CD80and CD86, also called B7-1 and B7-2 respectively, on antigen-presentingcells. CTLA4 transmits an inhibitory signal to T cells, whereas CD28transmits a stimulatory signal. Intracellular CTLA4 is also found inregulatory T cells and may be important to their function. T cellactivation through the T cell receptor and CD28 leads to increasedexpression of CTLA-4, an inhibitory receptor for B7 molecules.

In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4antibody (e.g., a human antibody, a humanized antibody, or a chimericantibody), an antigen binding fragment thereof, an immunoadhesin, afusion protein, or oligopeptide.

Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom)suitable for use in the present methods can be generated using methodswell known in the art. Alternatively, art recognized anti-CTLA-4antibodies can be used. For example, the anti-CTLA-4 antibodiesdisclosed in: U.S. Pat. No. 8,119,129; International Patent PublicationNos. WO 01/14424, WO 98/42752, and WO 00/37504 (CP675,206, also known astremelimumab; formerly ticilimumab); U.S. Pat. No. 6,207,156; Hurwitz etal., 1998; Camacho et al., 2004; and Mokyr et al., 1998 can be used inthe methods disclosed herein. The teachings of each of theaforementioned publications are hereby incorporated by reference.Antibodies that compete with any of these art-recognized antibodies forbinding to CTLA-4 also can be used. For example, a humanized CTLA-4antibody is described in International Patent Application Nos.WO2001014424, and WO2000037504, and U.S. Pat. No. 8,017,114; allincorporated herein by reference.

An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1,MDX-010, MDX-101, and Yervoy®) or antigen binding fragments and variantsthereof (see, e.g., WO 01/14424). In other embodiments, the antibodycomprises the heavy and light chain CDRs or VRs of ipilimumab.Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2,and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2 andCDR3 domains of the VL region of ipilimumab. In another embodiment, theantibody competes for binding with and/or binds to the same epitope onCTLA-4 as the above-mentioned antibodies. In another embodiment, theantibody has at least about 90% variable region amino acid sequenceidentity with the above-mentioned antibodies (e.g., at least about 90%,95%, or 99% variable region identity with ipilimumab).

Other molecules for modulating CTLA-4 include CTLA-4 ligands andreceptors such as described in U.S. Pat. Nos. 5,844,905, 5,885,796 andInternational Patent Application Nos. WO1995001994 and WO1998042752; allincorporated herein by reference, and immunoadhesins such as describedin U.S. Pat. No. 8,329,867, incorporated herein by reference.

4. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative, andpalliative surgery. Curative surgery includes resection in which all orpart of cancerous tissue is physically removed, excised, and/ordestroyed and may be used in conjunction with other therapies, such asthe treatment of the present embodiments, chemotherapy, radiotherapy,hormonal therapy, gene therapy, immunotherapy, and/or alternativetherapies. Tumor resection refers to physical removal of at least partof a tumor. In addition to tumor resection, treatment by surgeryincludes laser surgery, cryosurgery, electrosurgery, andmicroscopically-controlled surgery (Mohs' surgery).

Upon excision of part or all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection, or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

5. Other Agents

It is contemplated that other agents may be used in combination withcertain aspects of the present embodiments to improve the therapeuticefficacy of treatment. These additional agents include agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion,agents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers, or other biological agents. Increases inintercellular signaling by elevating the number of GAP junctions wouldincrease the anti-hyperproliferative effects on the neighboringhyperproliferative cell population. In other embodiments, cytostatic ordifferentiation agents can be used in combination with certain aspectsof the present embodiments to improve the anti-hyperproliferativeefficacy of the treatments. Inhibitors of cell adhesion are contemplatedto improve the efficacy of the present embodiments. Examples of celladhesion inhibitors are focal adhesion kinase (FAKs) inhibitors andLovastatin. It is further contemplated that other agents that increasethe sensitivity of a hyperproliferative cell to apoptosis, such as theantibody c225, could be used in combination with certain aspects of thepresent embodiments to improve the treatment efficacy.

IV. Kit

Also within the scope of the present disclosure are kits for detectingEGFR and/or HER2 exon 20 mutations, such as those disclosed herein. Anexample of such a kit may include a set of exon 20 mutation-specificprimer. The kit may further comprise instructions for use of the primersto detect the presence or absence of the specific EFGR and/or HER2 exon20 mutations described herein. The kit may further comprise instructionsfor diagnostic purposes, indicating that a positive identification ofEGFR and/or HER2 exon 20 mutations described herein in a sample from acancer patient indicates sensitivity to the tyrosine kinase inhibitorpoziotinib or afatinib or a structurally similar inhibitor. The kit mayfurther comprise instructions that indicate that a positiveidentification of EGFR and/or exon 20 mutations described herein in asample from a cancer patient indicates that a patient should be treatedwith poziotinib, afatinib, or a structurally similar inhibitor.

V. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—Identification of Drugs for Cancer Cells with EGFR or HER Exon20 Insertions

Clinical responses to TKIs were investigated in patients with tumorsharboring EGFR exon 20 insertions in the clinical database; and among280 patients with EGFR mutant NSCLC, 129 patients were identified withclassical EGFR mutations (exon 19 deletion, L858R, and L861Q) and 9patients with EGFR exon 20 insertions that were treated with singleagent erlotinib, gefitinib or afatinib. NSCLC patients with classicalEGFR mutations had a median PFS of 14 months, whereas patients with EGFRexon 20 insertions had a median PFS of only 2 months (p<0.0001, log ranktest; FIG. 1A). Of the 9 EGFR exon 20 insertion patients, OR wasobserved in only 1 patient harboring an S768del-insIL mutation whoreceived afatinib (FIG. 4A). This clinical data demonstrates the limitedactivity of the available EGFR TKIs in EGFR exon 20 insertion drivenNSCLC and validates that alternative treatment strategies are needed forthese specific tumors.

As an initial step in drug screening, 7 EGFR and 11 HER2 mutations wereexpressed in Ba/F3 cells. The locations of the EGFR and HER2 exon 20mutations are summarized in FIG. 1B. To assess which exon 20 mutationsof EGFR and HER2 are activating, Ba/F3 cell lines were screened for IL-3independent survival. It was found that all EGFR exon 20 insertionstested were activating mutations (FIG. 4B), 6 HER2 exon 20 mutationsand, L755P, located in exon 19, were activating mutations (FIG. 4C).Next, the sensitivity was tested for the exon 20 insertions to EGFR andHER2 TKIs that have undergone clinical evaluation including reversible(first generation), irreversible (second generation) and irreversiblemutant-specific TKIs (third generation), and then compared sensitivityto EGFR L858R, a classical sensitizing mutation. With the exception ofEGFR A763insFQEA, EGFR exon 20 insertions (n=6) were resistant to first(FIG. 1C, IC₅₀=3.3->10 μM), second (FIG. 1 d , IC₅₀=40-135 nM), andthird (FIG. 1 e , IC₅₀=103-850 nM) generation EGFR TKIs (FIG. 5 , Table1). In addition, HER2 exon 20 mutants (n=6) were resistant to first(FIG. 1F, IC₅₀=1.2-13 μM) and third (FIG. 1H, IC₅₀=114-505 nM)generation TKIs. Second generation TKIs did have some activity againstBa/F3 HER2 exon 20 mutant cell lines (FIG. 1G, IC₅₀=10-12 nM, FIG. 6 ,Table 1). Consistent with the drug screening, with the exception of EGFRA763insFQEA, which showed partial inhibition at lower doses, westernblotting demonstrated erlotinib and osimertinib did not significantlyinhibit p-EGFR2 in EGFR exon 20 insertion mutations, and onlysignificantly inhibited p-HER2 in HER2 exon 20 insertions mutants at 500nM (FIG. 7A-D).

TABLE 1 IC50 values of EGFR and HER2 exon 20 insertions with EGFR/HER2TKIs. Ave EGFR exon Ave HER2 exon 20 insertions 20 insertions (N = 6cell lines) (N = 6 cell lines) 1st gen Erlotinib 3,310 nM 3,250 nM TKIGefitinib >10,000 nM 12,900 nM Lapatinib — 1,190 nM L858R + Erlotinib17.0 nM 2nd gen Afatinib 39.9 nM 11.7 nM TKI Dacomitinib 61.1 nM 12.4 nMNeratinib 135 nM 10.4 nM L858R + Afatinib 0.876 nM  3rd gen Osimertinib103 nM 444 nM TKI Rociletinib 850 nM 505 nM Ibrutinib 143 nM 114 nMOlumtinib 204 nM 352 nM Nazartinib 198 nM 233 nM L858R/T790M + 9.00 nMOsimertinib

To investigate why exon 20 insertions are resistant to first and thirdgeneration EGFR TKIs, 3-D modeling was performed on the solved crystalstructures of EGFR D770insNPG with EGFR T790M and EGFR WT to visualizechanges within the drug binding pocket. The modeling revealed that EGFRexon 20 insertions are similar to T790M mutations in the alignment ofthe gatekeeper residue T790, which results in increased affinity to ATPand a reduced binding of first generation inhibitors, rendering thesemutations resistant to non-covalent inhibitors. In addition, HER2 exon20 insertions induce a constitutively active conformation, preventingthe binding of non-covalent HER2 inhibitor lapatinib, which binds toHER2 in the inactive conformation. Moreover, EGFR and HER2 exon 20insertions have a dramatic effect on the drug binding pocket. In silicomodeling of EGFR (FIG. 1I) and HER2 (FIG. 1J) exon 20 insertionsrevealed a significant shift of the α-c-helix into the drug bindingpocket (arrow) due to the insertions at the C-terminal end of theα-c-helix (FIG. 1J), forcing a ridged placement of the α-c-helix in theinward, activated position. In addition, 3-D modeling demonstrated asignificant shift of the P-loop into the drug binding pocket (FIG. 1I,1J) of both receptors. Together these shifts result in steric hindranceof the drug biding pocket from two directions in both EGFR and HER2 exon20 mutant proteins. Consistent with the above mentioned in vitrotesting, 3-D modeling supports the observation that afatinib inhibitsexon 20 insertions more effectively than osimertinib. Osimertinib has alarge terminal 1-methylindole group connected directly to a rigidpyrimidine core. This large inflexible group reduces the ability ofosimertinib to reach the C797 residue as effectively as afatinib in EGFRexon 20 insertions (FIG. 1I). Alternatively, afatinib has a smaller1-chloro-2-flurobenzene ring terminal group indirectly linked to aquinazoline core via a secondary amine group, enabling afatinib to fitinto the sterically hindered binding pocket. Moreover, steric hindranceprevents binding of osimertinib to HER2 A775insYVMA. Taken together, thein vitro data and in silico modeling indicate that small, flexiblequinazoline derivatives may be capable of targeting EGFR/HER2 exon 20insertions.

It was next sought to identify TKIs with enhanced activity against exon20 insertions. Poziotinib, like afatinib, also contains a small terminalgroup and a flexible quinazoline core. However, poziotinib has smallersubstituent groups linking the Michael Acceptor group to the quinazolinecore compared to afatinib and increased halogenation of the terminalbenzene ring compared to afatinib. This electron-rich moiety alsointeracts with basic residues of EGFR such as K745 to further stabilizeits binding. Therefore, poziotinib was tested in the Ba/F3 system. Invitro, poziotinib potently inhibited the growth of EGFR exon 20 mutantBa/F3 cell lines (FIG. 2A) and HER2 exon 20 mutant Ba/F3 cells (FIG.2B). Poziotinib had an average IC₅₀ value of 1.0 nM in EGFR exon 20mutant Ba/F3 cell lines making poziotinib approximately 100 times morepotent than osimertinib and 40 times more potent than afatinib in vitro.Moreover, poziotinib had an average IC₅₀ value of 1.9 nM in HER2 exon 20mutant Ba/F3 cell lines, making poziotinib 200 times more potent thanosimertinib and 6 times more potent than afatinib in vitro. Theseresults were validated by western blotting where poziotinib inhibitedphosphorylation of EGFR and HER2 at concentrations as low as 5 nM (FIG.2C, 8A). Furthermore, to validate that poziotinib sensitivity was notdue to level of expression of EGFR or HER2 mutants, expression of eachmutant was determined by ELISA then plotted against IC₅₀ values (FIG.8D). While no correlation was found between IC₅₀ and expression(R=−0.056, p=0.856), a correlation was found between poziotinibsensitivity and location of the mutation for EGFR (R=0.687, p=0.044)(FIG. 2D), suggesting that the further away the insertion is from theα-c-helix, the higher the IC₅₀. Interestingly, this correlation was notfound for HER2 exon 20 mutations which vary more in the size of theinsertion rather than the location (FIG. 8E). This correlation suggeststhat the precise location of the mutation has varying effects on thedrug binding pocket, contributing to the heterogeneity of drug responseseen. In addition, poziotinib effectively inhibited growth of patientderived cell lines CUTO14 (EGFR A767dupASV) and YUL0019 (EGFR N771delinsFH) with an average IC₅₀ value of 1.84 nM and 0.30 nM, respectively,which was 15 times more potent than afatinib for CUTO14 and more than100 times more potent than afatinib for YUL0019 (FIG. 2E, F). Westernblotting of CUTO14 cell line determined that there was significantinhibition of p-EGFR at 10 nM poziotinib treatment but p-EGFR was notsignificantly inhibited by afatinib until 1000 nM (FIG. 8B, C).

To determine the specificity of poziotinib to inhibit exon 20 mutantscompared to T790M mutants, the IC₅₀ values of afatinib, osimertinib,rociletinib, and poziotinib were compared in exon 20 mutants to the IC₅₀values of afatinib, osimertinib, rociletinib, and poziotinib in EGFRT790M mutant Ba/F3 cell lines. IC₅₀ values are displayed normalized tothe single EGFR T790M mutation, where values less than 1 indicatespecificity to exon 20 insertions compared to T790M (FIG. 2G). Whencompared to EGFR T790M mutants, EGFR exon 20 insertions were 65 timesmore sensitive to poziotinib. Moreover, EGFR exon 20 insertion mutationswere 1.4 times more resistant to afatinib, 5.6 times more resistant toosimertinib, and 24 times more resistant to rociletinib than EGFR T790Mmutants (FIG. 2G).

To examine why poziotinib, but not third generation TKIs such asosimertinib, selectively and potently inhibits exon 20 mutants comparedto T790M mutations, 3-D modeling was performed to determine how changesin the drug binding pocket affect drug binding. While osimertinib fitsinto the drug binding pocket of EGFR T790M mutant receptor (FIG. 2H), inexon 20 mutants, large changes (FIG. 2I) within the binding pocketsterically hinder the binding of third generation inhibitors. However,poziotinib is smaller and has greater flexibility allowing it to fitinto the sterically hindered exon 20 binding pocket (FIG. 2I). Moreover,3-D modeling of EGFR D770insNPG with poziotinib and afatinib suggestthat the shifted P-loop into the drug binding pocket causes poziotinibto bind more tightly into the drug binding pocket than afatinib.Calculations of structural modeling indicate that the free energy ofbinding (London ΔG) for poziotinib is lower than afatinib, indicatingstronger binding affinity of poziotinib. 3-D modeling of WT HER2 withosimertinib demonstrates that the binding pocket of WT HER2 is largerthan the binding pocket of HER2 A775insYVMA. Thus, poziotinib tightlybinds deep into the sterically hindered drug binding pocket of HER2A775insYVMA overcoming structural changes induced by exon 20 insertions.

The efficacy of poziotinib was tested in vivo using GEM models of EGFRand HER2 exon 20 insertion-driven NSCLC. Lung tumors were induced inpreviously described EGFR D770insNPG (Cho et al., 2013) and HER2A775insYVMA (Perera et al., 2009) mice, and animals orally receivedpoziotinib (10 mg/kg) or vehicle daily control for 4 weeks. Asdetermined by MRI, Poziotinib reduced tumor burden by 85% in EGFR exon20 GEMMs (FIGS. 3A,C) and 60% in HER2 exon 20 GEMMs (FIGS. 3B, D), ahigher level of inhibition than the 37% previously observed for afatinibin the identical GEM model. Representative MRI images of tumors beforeand after poziotinib are shown for both EGFR and HER2 GEMMs (FIGS. 3C,D). In both EGFR and HER2 GEM models, mice treated with 10 mg/kgpoziotinib demonstrated durable regression, without signs of progressionat 12 weeks (FIGS. 3E, F). In addition, poziotinib treatment (5 or 10mg/kg) completely reduced tumors by 14 days (>85% inhibition) in EGFRexon 20 insertions PDX model LU0387 (H773insNPH) (FIG. 3G).

To determine if poziotinib, like other irreversible inhibitors, bindscovalently at C797, Ba/F3 cell lines were generated with the C797Smutation observed in ˜30% of patients with osimertinib resistance(Thress et al., 2015). It was found that the C797S mutation inducedresistance to poziotinib with IC₅₀ value of >10 μM. Together theseexperiments suggested that poziotinib may be susceptible to similarmechanisms of acquired resistance as other third generation TKIs.

To validate the above findings, experiments were performed using abreast cancer cell line MCF10A with a HER2 G776del insVC. The cells weretreated with the different inhibitors at varying doses, and it was foundthat the breast cancer cell line is sensitive to poziotinib as seen inthe other cell lines tested (FIG. 10 ). Therefore, poziotinib can beused for the treatment of other cancers with exon 20 mutations.

Thus, it was found that exon 20 mutants exhibit de novo resistance tofirst, second, and third generation TKIs. Using 3-D modeling of EGFRD770insNPG and HER2 A775insYVMA poziotinib was identified as havingstructural features that could overcome changes within the drug bindingpocket induced by insertions in exon 20. Moreover, the predictedactivity of poziotinib was confirmed using in vitro and in vivo modelsdemonstrating the potent anti-tumor activity of poziotinib in cells withthese mutations.

Poziotinib was found to be approximately 40 times more potent thanafatinib and 65 times more potent than dacomitinib in EGFR exon 20mutants. Moreover, poziotinib was 6 times more potent that afatinib anddacomitinib in HER2 exon 20 mutants in vitro. Taken together, these dataindicate that although poziotinib shares a similar quinazoline backbonewith afatinib and dacomitinib, additional features of the kinaseinhibitor result in increased activity and relative specificity for EGFRexon 20 mutations compared with the more common T790M mutation.

The 3-D modeling suggests that the smaller size, increased halogenation,and flexibility of poziotinib give the inhibitor a competitive advantagein the sterically hindered drug binding pocket of exon 20 mutantEGFR/HER2. A negative correlation was observed between the distance ofthe mutation from the α-c-helix and drug sensitivity. This relationshipsuggests that the precise location of the mutation affects the drugbinding pocket and/or binding affinity of the TKI. Furthermore, the dataindicated that the size of the insertion also affects drug sensitivity.Furthermore, the patient derived cell line, YUL0019 (N771del insFH)which had a net gain of only one amino acid, was more sensitive toquinazoline based pan-HER inhibitors than cell lines with larger EGFRexon 20 insertions.

Example 2—Materials and Methods

Patient population and statistical analyses: Patients with EGFR mutantNSCLC enrolled in the prospectively collected MD Anderson Lung CancerMoon Shot GEMINI database were identified. EGFR mutation status wasdetermined using one of PCR-based next generation sequencing of panelsof 50, 134 or 409 genes used for routine clinical care. PFS wascalculated using the Kaplan Meier method. PFS was defined as time fromcommencement of EGFR TKI to radiologic progression or death. Restagingscans were obtained at 6-8 week intervals during treatment and wereretrospectively assessed according to the Response Evaluation Criteriain Solid Tumors (RECIST), version 1.1 to determine response rate inpatients with EGFR exon 20 insertion NSCLC.

Cell line generation and IL-3 deprivation: Ba/F3 cell line, was culturedin complete RPMI-1640 (R8758; Sigma Life Science) media supplementedwith L-glutamine, 10% heat inactivated FBS (Gibco), 1%penicillin/streptomycin (Sigma Life Science), and 10 ng/ml mouse IL-3(R&D systems) under sterile conditions. Stable cell lines were generatedby retroviral transduction of Ba/F3 cell line for 12 hours. Retroviruseswere generated by transfecting pBabe-Puro based vectors summarized inTable 2 (Addgene and Bioinnovatise) into the Phoenix 293T ampho packingcell line (Orbigen) using Lipofectamine 2000 (Invitrogen). 72 hoursafter transduction, 2 μg/ml puromycin (Invitrogen) was added to themedia. After 5 days of selection, cells were stained with FITC-HER2(Biolegend) or PE-EGFR (Biolegend) and sorted via FACS. Cell lines werethen grown in the absence of IL-3 for 15 days and cell viability wasdetermined every 3 days using the Cell Titer Glo assay (Progema).Resulting stable cell lines were maintained in complete RPMI-1640 mediadescribed above without IL-3. HCC827 and HCC4006 lung cancer cell lineswere obtained from ATCC and maintained in 10% RPMI media under sterileconditions. Cell line identity was confirmed by DNA fingerprinting viashort tandem repeats using the PowerPlex 1.2 kit (Promega).Fingerprinting results were compared with reference fingerprintsmaintained by the primary source of the cell line. All cell lines werefree of mycoplasma. To generate erlotinib resistant cell lines, HCC827and HCC4006 (both EGFR mutant) cells were cultured with increasingconcentrations of erlotinib until resistant variants emerged.

TABLE 2 Vector used to generate stable cell lines. Name Mutation VendorEGFR c.2290_2291insTCCAGGAAGCCCreated from Bioinnovatise from pBabe-puro- A763insFQEA T (SEQ ID NO: 2)EGFR WT from Addgene (#11011) (SEQ ID NO: 1) EGFRc.2302_2303insGCCAGCGTG Purchased from Addgene (#32066) A767insASV EGFRc.2303_2304dupAGCGTGGAC Created from Bioinnovatise from pBabe-puro-S768dupSVD EGFR WT from Addgene (#11011) EGFR c.2308_2309insCCAGCGTGGCreated from Bioinnovatise from pBabe-puro- V769insASVEGFR WT from Addgene (#11011) EGFR c.2310_2311insAACCCCGGCPurchased from Addgene (#11016) D770insNPG EGFR c.2311_2312insGCGTGGACACreated from Bioinnovatise from pBabe-puro- D770insSVDEGFR WT from Addgene (#11011) EGFR c.2319_2320insAACCCCCACCreated from Bioinnovatise from pBabe-puro- H773insNPHEGFR WT from Addgene (#11011) EGFR Purchased from Addgene (#32070) T790MEGFR Purchased from Addgene (#32073) T790M L858R EGFRPurchased from Addgene (#32072) T790M Ex19del EGFR T790M c.2389T > ACreated from Bioinnovatise from pBabe-puro- L858R C797SEGFR L858R/T790M from Addgene (#32073) EGFR T790M c.2389T > ACreated from Bioinnovatise from pBabe-puro- Ex19del C797SEGFR Del1/T790M from Addgene (#32072) HER2 c.929C > TPurchased from Addgene (#40991) S310F HER2 c.929C > APurchased from Addgene (#40992) S310Y HER2 c.931T > CPurchased from Addgene (#40980) C311R HER2 c.2263_2264delinsCCCreated by Bioinnovatise from pBabe-puro L755PHER2 WT from Addgene (#40978) HER2 c.2323-2324insTTTPurchased from Addgene (#40979) A775insV G776C HER2c.2323_2324insTATGTCATGGCT Purchased from Addgene (#40982) A775insYVMA(SEQ ID NO: 4) (SEQ ID NO: 3) HER2 c.2327G > TCreated by Bioinnovatise from pBabe-puro G776VHER2 WT from Addgene (#40978) HER2 c.2326G > T,Created by Bioinnovatise from pBabe-puro G776C V777insVc.2331_2332insTGT HER2 WT from Addgene (#40978) HER2 c.2327delinsTTGTCreated by Bioinnovatise from pBabe-puro G776del insVVHER2 WT from Addgene (#40978) HER2 c.2326_2328insTCTCreated by Bioinnovatise from pBabe-puro G776del ins VCHER2 WT from Addgene (#40978) HER2 c.2339_2340insTGGCTCCCCCreated by Bioinnovatise from pBabe-puro P780insGSPHER2 WT from Addgene (#40978)

Cell Viability Assay and IC₅₀ Estimation: Cell viability was determinedusing the Cell Titer Glo assay (Promega). Cells were collected fromsuspension media, spun down at 300×g for 5 minutes and re-suspended infresh RPMI media and counted using a Countess automated cell counter andtrypan blue (Invitrogen). 1500 cells per well were plated in 384-wellplates (Greiner Bio-One) in technical triplicate. Cells were treatedwith seven different concentrations of inhibitors in serial three-folddiluted TKIs or vehicle alone at a final volume of 40 μL per well. After72 hours, 11 μL of Cell Titer Glo was added to each well. Plates wereshaken for 10 minutes, and bioluminescence was determined using aFLUOstar OPTIMA multi-mode micro-plate reader (BMG LABTECH).Bioluminescence values were normalized to DMSO treated cells, andnormalized values were plotted in GraphPad Prism using non-linearregression fit to normalized data with a variable slope. IC₅₀ valueswere calculated by GraphPad Prism at 50% inhibition. Each experiment wasreplicated 3 times unless indicated.

Tyrosine Kinase Inhibitors: Lapatinib, afatinib, dacomitinib, AZD9291,CO-1686, EGF816, ibrutinib, and HM781-36B were purchased from SelleckChemical. Erlotinib and gefitinib were obtained from the institutionalpharmacy at The University of Texas MD Anderson Cancer Center. BI-694was provided by Boehringer-Ingelheim. All inhibitors were dissolved inDMSO at a concentration of 10 mM and stored at −80° C.

3-D modeling: The structure of EGFR D770insNPG protein was retrieved(Protein Data Bank entry code: 4LRM) and used it as a template to buildour molecular 3-D structural model of EGFR D770insNPG. HER2 A775insYVMAwas built using the previously published model in Shen et al. Thehomology models were built using MODELLER 9v6 and further energeticallyminimized using Molecular Operating Environment software package(Chemical Computing Group, Montreal, Canada). Molecular docking of TKIsinto exon 20 mutant EGFR and HER2 were performed using GOLD softwarewith default parameters unless otherwise noted. No early termination wasallowed in the docking process. Restraints were used to model thecovalent bond formations between receptors and inhibitors. Theflexibility of residues within the binding pocket was addressed usingGOLD software. Figures demonstrating interactions between EGFR/HER2 andinhibitors were visualized using PYMOL.

Western Blotting of Ba/F3 mutants: For Western blotting, cells werewashed in phosphate-buffered saline and lysed in protein lysis buffer(ThermoFisher) and protease inhibitor cocktail tablets (Roche). Protein(30-40 μg) was loaded into gels purchased from BioRad. BioRad semi-drytransfer was used and then probed with antibodies against pEGFR (#2234),EGFR (#4267), pHER2 (#2247), HER2 (#4290) (1:1000; Cell Signaling).Blots were probed with antibodies against β-actin (Sigma-Aldrich,#A2228) or vinculin (Sigma-Aldrich, #V4505) as a loading control, andexposed using SuperSignal West Pico Chemiluminescent Substrate(ThermoFisher) and BioRad's ChemiDoc Touch Imaging System orradiographic film. Representative images are shown of two separateprotein isolations and blots run in duplicate. Quantification of westernblotting was completed in Photoshop and calculated as (background meanintensity−sample mean intensity) (number of pixels)=band intensity.Samples were normalized first to loading control (β-actin or vinculin),then normalized to DMSO and graphed in GraphPad Prism. Significance fromDMSO was calculated in GraphPad Prism.

ELISA and correlation of Ba/F3 mutants: Protein was harvested from theparental Ba/F3 cell line and each of the Ba/F3 exon 20 mutants found tobe activating mutations as described above. ELISA was performed asdescribed by the manufacture instructions for total EGFR (Cellsignaling, #7250) and total HER2 (Cell Signaling, #7310). Relativeexpression determined by ELISA was plotted against IC₅₀ valuescalculated as described above. Pearson correlations and p-values weredetermined by GraphPad Prism.

Patient Derived Cell line studies: CUTO14 cells were generated from thepleural effusion of a patient with lung adenocarcinoma followinginformed consent using previously described culture methods (Davies etal., 2013). Cell lines were treated with the indicated doses of afatinibor poziotinib for 72 hours and cell viability was determined by MTSassay (Promega). IC50 was calculated as previously described (n=3).Western blotting with patient derived cell lines was completed aspreviously described (Hong et al., 2007) (n=3). Cells were treated for 2hours with indicated doses of afatinib or poziotinib. All antibodieswere purchased from Cell Signaling Technology with the exception oftotal EGFR (BD Transduction Laboratories) and GAPDH (Calbiochem).

The YUL0019 cell line was established from malignant pericardial fluidobtained from a patient with advanced adenocarcinoma of the lung underan IRB-approved protocol. The cell line was cultured in RPMI+L-glutamine(Corning), supplemented with 10% heat-inactivated fetal bovine serum(Atlanta Biologicals) and 1% penicillin/streptomycin (Corning). Toconfirm the presence of the EGFR mutation, RNA was extracted from cellpellet using the RNeasy mini kit (Qiagen #74104) according tomanufacturer's instructions. cDNA was synthesized using the SuperscriptIII First-Strand cDNA Synthesis Kit (Invitrogen #18080-051) and used asa template to amplify EGFR. PCR product was sequenced by Sangersequencing using the following primers: EGFR-2080F: CTTACACCCAGTGGAGAAGC (SEQ ID NO:5) and EGFR-2507R ACCAAGCGACGGTCCTCCAA (SEQ IDNO:6). Forward and reverse sequence tracings were manually reviewed. Thevariant detected in the patient-derived cell line was a complexinsertion in exon 20 of EGFR (N771delinsFH) leading to the replacementof amino acid asparagine at position 771 by two amino acids,phenylalanine and histidine. Cell viability and IC₅₀ estimation wasperformed as described above.

Patient Derived Xenograft (PDX) studies: LU0387 PDX experiments werecompleted by Crown BioSciences. Briefly, tumor fragments from EGFRH773insNPH expressing tumors were inoculated into 5-6 week old femalenu/nu nude mice. When tumors reached 100-200 mm³ mice were randomizedinto 3 groups: 5 mg/kg poziotinib, 10 mg/kg poziotinib, or vehiclecontrol (20% PEG-400, 3% Tween-80 in dH₂O). Tumor volumes and bodyweight were measured twice weekly. Mice receiving 5 mg/kg poziotinibreceived drug for 4-5 days then were on dosing holiday for 4 days thenreceived 4 additional days of dosing. Mice were then observed for 2additional days without dosing. Mice receiving 10 mg/kg poziotinibreceived drug for 3-4 days then were observed for 10 days withoutdosing. Mice humanly euthanized for events unrelated to tumor burdenwere excluded from final analysis.

Genetically Engineered Mouse Model (GEMM) studies: EGFR D770insNPG andHER2 A775insYVMA GEMMs were generated as previously described (Perera etal., 2009; Cho et al., 2013). Mice were handled in accordance with GoodAnimal Practices as defined by the Office of Laboratory Animal Welfareand done in with approval from Dana-Farber Cancer InstituteInstitutional Animal Care and Use Committee (Boston, Mass.). Mice werefed a continuous doxycycline diet from 6 weeks of age. Tumor volume wasdetermined by MRI as previously described (Perera et al., 2009; Cho etal., 2013). Mice with equal initial tumor volume were non-blindlyrandomized to vehicle and 10 mg/kg poziotinib daily upon obvious tumorformation determined by MRI. Mice humanly euthanized for eventsunrelated to tumor burden were excluded from final analysis.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1-36. (canceled)
 37. A method of treating cancer in a subject comprisingadministering an effective amount of poziotinib to the subject, whereinthe subject has a tumor that has been determined to have one or moreHER2 exon 20 mutations, wherein the one or more HER2 exon 20 mutationscomprise one or more point mutations, insertions, and/or deletions of1-6 amino acid between amino acids 770-785, wherein the one or more HER2exon 20 mutations are at residue A775, S779, and/or P780, and/or whereinthe one or more HER2 exon 20 mutations are selected from the groupconsisting of A775insV G776C, A775insYVMA, G776V, G776C V777insV, G776CV777insC, G776del insVV, G776del insVC, and P780insGSP.
 38. (canceled)39. (canceled)
 40. The method of claim 37, wherein the HER exon 20mutation is further defined as a HER2 exon 20 insertion mutation. 41.The method of claim 40, wherein the HER exon 20 insertion mutation isA775insYVMA.
 42. The method of claim 37, further comprisingadministering an mTOR inhibitor.
 43. The method of claim 42, wherein themTOR inhibitor is rapamycin, temsirolimus, everolimus, ridaforolimus orMLN4924.
 44. The method of claim 42, wherein the mTOR inhibitor iseverolimus.
 45. The method of claim 42, wherein the poziotinib and/orthe mTOR inhibitor are administered intravenously, subcutaneously,intraosseously, orally, transdermally, in sustained release, incontrolled release, in delayed release, as a suppository, orsublingually.
 46. (canceled)
 47. (canceled)
 48. (canceled)
 49. Themethod of claim 37, further comprising administering an additionalanti-cancer therapy.
 50. The method of claim 37, wherein the additionalanti-cancer therapy is chemotherapy, radiotherapy, gene therapy,surgery, hormonal therapy, anti-angiogenic therapy or immunotherapy. 51.The method of claim 37, wherein the cancer is oral cancer, oropharyngealcancer, nasopharyngeal cancer, respiratory cancer, urogenital cancer,gastrointestinal cancer, central or peripheral nervous system tissuecancer, an endocrine or neuroendocrine cancer or hematopoietic cancer,glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma,brain cancer, oropharyngeal cancer, nasopharyngeal cancer, renal cancer,biliary cancer, pheochromocytoma, pancreatic islet cell cancer,Li-Fraumeni tumors, thyroid cancer, parathyroid cancer, pituitarytumors, adrenal gland tumors, osteogenic sarcoma tumors, multipleneuroendocrine type I and type II tumors, breast cancer, lung cancer,head and neck cancer, prostate cancer, esophageal cancer, trachealcancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer,ovarian cancer, uterine cancer, cervical cancer, testicular cancer,colon cancer, rectal cancer or skin cancer.
 52. The method of claim 37,wherein the cancer is non-small cell lung cancer.
 53. The method ofclaim 37, wherein the subject is human.
 54. A pharmaceutical compositioncomprising poziotinib or afatinib for use in a subject determined tohave one or more HER2 exon 20 mutations, wherein the one or more HER2exon 20 mutations comprise one or more point mutations, insertions,and/or deletions of 1-6 amino acids between amino acids 770-785, whereinthe one or more HER2 exon 20 mutations are at residue A775, S779, and/orP780, and/or wherein the one or more HER2 exon 20 mutations are selectedfrom the group consisting of A775insV G776C, A775insYVMA, G776CV777insC, G776del insVV, G776del insVC, and P780insGSP.
 55. Thecomposition of claim 54, wherein the HER2 exon 20 mutation is furtherdefined as a HER2 exon 20 insertion mutation.
 56. (canceled) 57.(canceled)
 58. (canceled)
 59. A method of predicting a response topoziotinib alone or in combination with an anti-cancer therapy in asubject having a cancer comprising detecting an HER2 exon 20 mutationselected from the group consisting of A775insV G776C, A775insYVMA, G776CV777insC, G776del insVV, G776del insVC, and P780insGSP in a genomicsample obtained from said subject, wherein if the sample is positive forthe presence of the HER2 exon 20 mutation, then the patient is predictedto have a favorable response to the poziotinib or afatinib alone or incombination with an anti-cancer therapy. 60-79. (canceled)
 80. Themethod of claim 37, wherein the one or more HER2 exon 20 mutationscomprise one or more point mutations, insertions, and/or deletions of3-18 between amino acids 770-785.
 81. The method of claim 37, whereinthe one or more HER2 exon 20 mutations are at residue A775, S779, and/orP780.
 82. The method of claim 37, wherein the one or more HER2 exon 20mutations are selected from the group consisting of A775insV G776C,A775insYVMA, G776V, G776C V777insV, G776C V777insC, G776del insVV,G776del insVC, and P780insGSP